Restoration Of Fluorescence Research Articles

Liposome: a tool to raise the Cherenkov radiation yield and to restore fluorophore properties in aqueous media.

Liposomes used for the study were prepared and carefully characterized multiple times until all batches indicated the same characterization data (DOPC/cholesterol derivative (1 : 1.15 mol%), 14 mg(DOPC) mL-1, dDLS = 130 nm, 2 × 1011 liposome per nm3 of prepared batch, polydispersity index PDI = 0.1). The study shows that such a liposome suspension raises the yield in Cherenkov Radiation (CR) by 1.6-fold when in presence of [68Ga]-GaCl3, an efficient CR emitter (beta particle energy Eβ+ = 1800 KeV). Also, liposomes were found to prevent aggregation of a water-soluble phthalocyanine-pyranine PcPy4 dyad upon encapsulation, leading to its spectacular fluorescence restoration. Altogether, upon efficient 68Ga-radiolabelling of NODAGA-chelate immobilized at the liposome surface (99% radiolabelling yield, radio-TLC showed) encapsulating PcPy4 dyad (Caverage = 97 μM, Clumen = 300 μM), subsequent Cherenkov Radiation Energy Transfer (CRET) at the dyad antenna occurred. Internal energy transfers and fluorescence emission from the emitter dyad led to a 2.6-fold raise in radiance measured in the near-infrared (NIR) window (i.e. ca. 400 nm pseudo-Stokes shift). A similar raise in radiance was measured in the green window when encapsulation was achieved with eosin at the same rate. Liposome were found to be stable in PBS over 7 days regardless of the encapsulated fluorophore (no raise in dDLS diameter, no release of encapsulated dyes measured after Sephadex and FPLC repurification sequence), with some decay over 22 hours in a PBS/fetal calf serum mixture (1 : 1 vol.).

Towards the Development of an Optical Biosensor for the Detection of Human Blood for Forensic Analysis.

Blood is a common biological fluid in forensic investigations, offering significant evidential value. Currently employed presumptive blood tests often lack specificity and are sample destructive, which can compromise downstream analysis. Within this study, the development of an optical biosensor for detecting human red blood cells (RBCs) has been explored to address such limitations. Aptamer-based biosensors, termed aptasensors, offer a promising alternative due to their high specificity and affinity for target analytes. Aptamers are short, single-stranded DNA or RNA sequences that form stable three-dimensional structures, allowing them to bind to specific targets selectively. A nanoflare design has been employed within this work, consisting of a quenching gold nanoparticle (AuNP), DNA aptamer sequences, and complementary fluorophore-labelled flares operating through a fluorescence resonance energy transfer (FRET) mechanism. In the presence of RBCs, the aptamer-flare complex is disrupted, restoring fluorescence and indicating the presence of blood. Two aptamers, N1 and BB1, with a demonstrated binding affinity to RBCs, were selected for inclusion within the nanoflare. This study aimed to optimise three features of the design: aptamer conjugation to AuNPs, aptamer hybridisation to complementary flares, and flare displacement in the presence of RBCs. Fluorescence restoration was achieved with both the N1 and BB1 nanoflares, demonstrating the potential for a functional biosensor to be utilised within the forensic workflow. It is hoped that introducing such an aptasensor could enhance the forensic workflow. This aptasensor could replace current tests with a specific and sensitive reagent that can be used for real-time detection, improving the standard of forensic blood analysis.

A Novel Carbon Dot-Bromothymol Blue System for Ratiometric Colorimetric-Fluorometric Sensing of Glutathione in Urine: A Smartphone-Compatible Approach.

This study presents a novel dual-modal approach for glutathione (GSH) detection using blue and yellow dual-emission carbon dots (BY-CDs) and bromothymol blue (BTB) at pH 8.0. The method employs both colorimetric and fluorometric detection modes, offering a new perspective on GSH quantification. BTB's blue coloration induces selective fluorescence quenching of the CDs' 610nm emission peak, with minimal effect on the 445nm peak. Upon GSH addition, the quinonoid structure (blue color) of BTB transforms to its benzenoid form (yellow color). This transformation triggers fluorescence restoration at 610nm and significant quenching at 445nm, enabling ratiometric fluorescence analysis (F610/F445). Concurrently, colorimetric detection is also ratiometric, based on measuring the ratio between the emerging yellow color peak (435nm) and the decreasing blue color peak (618nm) (A435/A618). The state-of-the-art aspect of this detection method lies in the strategic choice of dual-emission CDs and a dye with distinct absorption spectra that closely match the emission spectra of the CDs. This unique combination allows for dual detection with opposite responses in the two detection modes, enhancing selectivity and reliability. The probe was thoroughly characterized, and its detection mechanism was elucidated using various spectroscopic techniques. The method shows excellent linearity, a broad detection range, and low detection limits for both fluorometry (0.02 - 10.0μM, 5.88nM) and colorimetry (1.0 - 35.0μM, 301.25nM). Additionally, a smartphone-based platform was developed for colorimetric GSH determination, enhancing the method's potential for on-site analysis. The assay's practicality was validated through successful application to human urine samples, yielding excellent recovery values (97.33% to 99.13%).

Fluorescence switching sensor for sensitive detection of curcumin/Mn2+ using biomass carbon quantum dots: A combination of experimental and theoretical insights

Compared to high-performance liquid chromatography, capillary electrophoresis, and electrochemical analysis, fluorescent carbon quantum dots (CQDs)derived from biomass materials have garnered significant attention due to their non-toxic, eco-friendly, and renewable advantages. In this research, CQDs-NH2 was synthesized in one step by hydrothermal method using banana peel as biomass carbon source and ethylenediamine (EDA) as dopant. The blue fluorescence of CQDs-NH2 was found to be quenched by the addition of curcumin through the synergistic effect of static quenching and internal filtering effect (IFE). The response to curcumin showed good linearity in the concentration range of 1–15 μM with a limit of detection (LOD) of 18.56 nM. The fluorescence of CQDs-NH2 was partially restored in the presence of curcumin and Mn2+, leading to the establishment of a curcumin@CQDs-NH2 switch sensor for the detection of Mn2+ in the linear range of 5–60 μM with a LOD of 20.35 nM. Furthermore, density functional theory (DFT) calculations suggest that the interaction between Mn2+ and curcumin is stronger than the interaction between Mn2+ and functionalized CQDs (f-CQDs), elucidating the introduction of Mn2+ disrupts the interaction between curcumin and CQDs-NH2, leading to the restoration of CQDs-NH2 fluorescence. Finally, the CQDs-NH2 fluorescence switch sensor demonstrated high accuracy and precision in liquid food and water quality testing with recoveries ranging from 95.19 % to 105.54 %.

Enzyme-aided amplification strategy for sensitive detection of methamphetamine based on fluorescence aptamer sensor.

Methamphetamine (METH) abuse poses a serious risk to human health and social stability. It is critical to develop sensitive and selective methods for detecting METH. Here, we develop a fluorescence aptamer sensor to detect METH based on DNA exonuclease III (Exo III), graphene oxide (GO), and FAM-labeled aptamer. First, the sensor used GO's strong binding capacity to adsorb and quench the fluorescence of the aptamer attached to GO surface. When METH was added to the system, the formation of stable complex for aptamer and METH dissociated from the surface of GO, leading to a fluorescence restoration. Then, the fluorescence signal was further amplified by using Exo III to liberate target METH for cyclic hybridization. And the gel electrophoresis experiment further verified the reliability of this strategy. This aptamer sensor exhibited a low detection limit (0.52nM) and excellent selectivity under optimal conditions. Notably, this sensor has been successfully validated in the detection of METH in urine and saliva samples, exhibited commendable recovery (94.00-104.65%). Its benefits include facile, sensitive, and rapid. Expected to be used in practical METH detection.

Molecular imprinting film and turn-on fluorescence probe working in tandem for Salmonella Typhimurium detection

Salmonella is one of the most common pathogens that cause foodborne illnesses. Rapid qualitative and quantitative detection of Salmonella Typhimurium (S. Typhimurium) is important for human health. In this study, a Förster resonance energy transfer (FRET)-based platform was proposed to activate fluorescence sensing with a bacteria-imprinted film to facilitate S. Typhimurium detection. Imprinted films with surface cavity sites that match the surface cavity sites of S. Typhimurium was prepared. The modification of the films using 1H,1H,2H,2H-perfluorooctyltriethoxysilane (POTS), weakened the hydrophobicity of the films, inhibiting the nonspecific adsorption of nontarget bacteria. Copper nanoclusters and gold nanoparticles, which are good fluorescent molecular donors and acceptors, respectively, can be used to prepare fluorescence quenching-recovery probes based on FRET. The competitive binding of the bacteria to the probes enabled the restoration of fluorescence, facilitating bacteria quantification based on the fluorescence intensity. The optimized fluorescent sensor showed a linear response to S. Typhimurium concentrations in the range of 10–107 CFU/mL with a detection limit of 17.38 CFU/mL. Thus, the proposed label-free fluorescent sensor is sensitive to S. Typhimurium, showing potential applications in the detection of this pathogen as well as other foodborne pathogens in food samples.

Fluorescence Quenching Effect of a Highly Active Nitroxyl Radical on 7-amino-4-methylcoumarin and Glutathione Sensing.

Nitroxyl radical compounds, such as 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), are stable radical compounds with a variety of unique characteristics, including fluorescence quenching. In this study, we investigated the fluorescence quenching effect of nortropine N-oxyl (NNO), which is a highly active nitroxyl radical that is more active than TEMPO in oxidation catalysis. The fluorescence intensity of 7-amino-4-methylcoumarin (AMC) was quenched by NNO and TEMPO to 5% and 95% of the initial fluorescence intensity, respectively, indicating highly efficient quenching by NNO. In addition, we used this reaction to measure glutathione concentration. The quenching effect of NNO was abrogated by the chemical reaction with glutathione, resulting in restoration of AMC fluorescence. This response was observed at glutathione concentrations from 10 µM to 1 mM, and good calibration curves were obtained from 10 to 250 µM.

FcCe-MOF-NH2-based colorimetric and fluorometric dual-mode detection of sulfide ions

Sulfide ions, commonly present as toxic anions in industrial processes, require the creation of a convenient and reliable detection platform. In this study, we developed a dual-mode colorimetric and fluorometric method to detect sulfide ions using the cerium-ferrocene bimetal-organic framework (MOF), i.e., FcCe-MOF-NH2, which was synthesized by a common solvo-thermal approach. The FcCe-MOF-NH2 promises the peroxidase-like activity and blue emission peaked at 453 nm. In the presence of H2O2, the FcCe-MOF-NH2 catalyzed 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation, yielding the blue oxidized product (oxTMB), while the fluorescence peak at 453 nm was suppressed due to the inner filter effect (IFE). The Km values of FcCe-MOF-NH2 for TMB and H2O2 were 0.099 mM and 0.179 mM, respectively, showing its good affinity with the substrates. Due to a unique redox reaction between H2O2 and S2−, the peroxidase-like activity of FcCe-MOF-NH2 was markedly suppressed, leading to fluorescence restoration. Hence, the colorimetric and fluorometric dual-mode detection of S2− was realized, exhibiting a linear range of 0.5–100 μM and 0.2–60 μM with a detection limit of 0.43 μM and 0.078 μM, respectively. The developed platform was successfully applied to the detection of S2− in actual water samples.

Detection of Arsenic(V) by Fluorescence Sensing Based on Chlorin e6-Copper Ion.

The high toxicity of arsenic (As) can cause irreversible harm to the environment and human health. In this study, the chlorin e6 (Ce6), which emits fluorescence in the infrared region, was introduced as the luminescence center, and the addition of copper ion (Cu2+) and As(V) provoked a regular change in fluorescence at 652 nm, whereas that of As(III) was 665 nm, which was used to optionally detect Cu2+, arsenic (As(III), and As(V)). The limit of detection (LOD) values were 0.212 μM, 0.089 ppm, and 1.375 ppb for Cu2+, As(III), and As(V), respectively. The developed method can be used to determine Cu2+ and arsenic in water and soil with good sensitivity and selectivity. The 1:1 stoichiometry of Ce6 with Cu2+ was obtained from the Job plot that was developed from UV-visible spectra. The binding constants for Cu2+ and As(V) were established to be 1.248 × 105 M-1 and 2.35 × 1012 M-2, respectively, using B-H (Benesi-Hildebrand) plots. Fluorescence lifetimes, B-H plots, FT-IR, and 1H-NMR were used to postulate the mechanism of Cu2+ fluorescence quenching and As(V) fluorescence restoration and the interactions of the two ions with the Ce6 molecule.

Simple, sensitive, colorimetric detection of pyrophosphate via the analyte-triggered decomposition of metal-organic frameworks regulating their adaptive multi-color Tyndall effect.

This paper describes initially the application of the Tyndall effect (TE) of metal-organic framework (MOF) materials as a colorimetric signaling strategy for the sensitive detection of pyrophosphate ion (PPi). The used MOF NH2-MIL-101(Fe) was prepared with Fe3+ ions and fluorescent ligands of 2-amino terephthalic acid (NH2-BDC). The fluorescence of NH2-BDC in MOF is quenched due tothe ligand-to-metal charge transfer effect, while the NH2-MIL-101(Fe) suspension shows a strong TE. In the presence of PPi analyte, the MOFs will undergo decomposition because of the competitive binding of Fe3+ by PPi over NH2-BDC, resulting in a significant decrease in the TE signal and fluorescence restoration from thereleased ligands. The results demonstrate that the new method only requires a laser pointer pen (for TE creation) and a smartphone (for portable quantitative readout) to detect PPi in a linear concentration range of 1.25-800 μM, with a detection limit of ~210 nM (3σ) which is ~38 times lower than that obtained from traditional fluorescence with a spectrophotometer (linear concentration range, 50-800 µM; detection limit, 8.15 µM). Moreover, the acceptable recovery of PPi in several real samples (i.e., pond water, black tea, and human serum and urine) ranges from 97.66 to 119.15%.

Carbon dots and covalent organic frameworks based FRET immunosensor for sensitive detection of Escherichia coli O157:H7

The combination of carbon dots (CDs) with covalent organic frameworks (COFs) was used to design an innovative sensor based on fluorescence resonance energy transfer (FRET) for the detection of Escherichia coli O157:H7 (E. coli O157:H7) in food samples. Carbon dots were used as fluorescence donors, covalent organic frameworks as fluorescence acceptors. The antibody (Ab) specific to E. coli O157:H7 was used to form a CD-Ab-COF immunosensor by linking CDs and COFs. The antibody was specifically bound with E. coli O157:H7, which caused the connection between CDs and COFs to be interrupted, and the carbon dots exhibited fluorescence restoration. The sensor exhibited a linear detection range spanning from 0 to 106 CFU/mL, with the limit of detection (LOD) of 7 CFU/mL. The analytical performance of the developed immunosensor was evaluated using spiked food samples with different concentrations of E. coli O157:H7, validating the capability of assessing risks in food testing.

Aptamer-based fluorescent sensor for highly sensitive detection of methamphetamine.

The construction of a fluorescence aptamer sensor was achieved by employing the fundamental principle of fluorescence resonance energy transfer. By employing molecular modeling technologies to identify the binding site, the high-affinity aptamer APT-40nt was derived from the whole sequence and utilized on the graphene oxide (GO) fluorescent platform for the purpose of achieving a highly sensitive detection of methamphetamine (METH). The aptamer tagged with fluorescein (FAM) dye undergoes quenching in the presence of GO due to π-stacking interaction. With the addition of the target, the aptamer that has been tagged was detached from the GO surface, forming a stable complex with METH. This process resulted in fluorescence restoration of the system, and the degree of fluorescence restoration was proportional to METH concentration in the linear range of 1-50 and 50-200 nM. Notably, under optimized conditions, the detection limit of this aptasensor was as low as 0.78 nM, which meets the detection limit requirements of METH detection in saliva and urine in some countries and regions. Moreover, other common illicit drugs and metabolites had minimizing interference with the determination. The established aptasensor, therefore, has been successfully applied to detect METH in saliva and urine samples and exhibited satisfactory recoveries (87%-111%). This aptasensor has the advantages of low detection limit, excellent selectivity, ease of operation, and low cost, providing a promising strategy for on-site detection of METH in saliva and urine.

A facile fluorescence Eu MOF sensor for ascorbic acid and ascorbate oxidase detection.

In this work, a facile fluorescence Eu3+-based metal-organic framework (Eu MOF) sensor for ascorbic acid (AA) and ascorbate oxidase (AAO) detection was developed. The fluorescence of the Eu MOF could be effectively quenched by Ce3+ but not by Ce4+ at an appropriate concentration, and thus, when the reductant AA was added into the solution containing Ce4+, Ce4+ was chemically reduced to Ce3+, which induced the decreased fluorescence signal of the Eu MOF. However, when AAO was introduced, AA was effectively oxidized to dehydroascorbic acid (DHAA) under the catalysis of AAO, and thus, Ce4+ could not be reduced, resulting in the fluorescence restoration of the Eu MOF. Hence, the concentration of AA and AAO could be determined by the fluorescence decrease and restoration of the Eu MOF. The fluorescent platform showed high sensitivity with a limit of detection of 0.32 μM for AA and 1.18 U L-1 for AAO, respectively. Moreover, the proposed method was successfully applied for AA and AAO determination in real samples, indicating great potential for biomedical application in complex matrices.

A label-free ratiometric fluorescent aptasensor based on a peroxidase-mimetic multifunctional ZrFe-MOF for the determination of tetrodotoxin.

A Fe/Zr bimetal-organic framework (ZrFe-MOF) is utilized to establish a ratiometric fluorescent aptasensor for the determination of tetrodotoxin (TTX). The multifunctional ZrFe-MOF possesses inherent fluorescence at 445nm wavelength, peroxidase-mimetic activity, and specific recognition and adsorption capabilities for aptamers, owing to its organic ligand, and Fe and Zr nodes. The peroxidation of o-phenylenediamine (OPD) substrate generates fluorescent 2,3-diaminophenazine (OPDox) at 555nm wavelength, thus quenching the inherent fluorescence of ZrFe-MOF because of the fluorescence resonance energy transfer (FRET) effect. TTX aptamers, which are absorbed on the material surface without immobilization or fluorescent labeling, inhibit the peroxidase-mimetic activity of ZrFe-MOF. It causes the decreased OPDox fluorescence at 555nm wavelength and the inverse restoration of ZrFe-MOF fluorescence at 445nm wavelength. With TTX, the aptamers specifically bind to TTX, triggering rigid complex release from ZrFe-MOF surface and reactivating its peroxidase-mimetic activity. Consequently, the two fluorescence signals exhibit opposite changes. Employing this ratiometric strategy, the determination of TTX is achieved with a detection limit of 0.027ng/mL and a linear range of 0.05-500ng/mL. This aptasensor also successfully determines TTX concentrations in puffer fish and clam samples, demonstrating its promising application for monitoring trace TTX in food safety.

A dual-mode aptasensor based on freeze–thaw induced aggregation of gold nanoparticles and FRET for Staphylococcus aureus detection

The foodborne pathogen Staphylococcus aureus (S. aureus), known for its high virulence, presents a substantial risk to food safety. In this study, we proposed a detection platform utilizing the aptamer-induced aggregation of gold nanoparticles (AuNPs) through freeze–thaw and the fluorescence resonance energy transfer (FRET) between FITC and AuNPs. This platform offered an efficient means of identifying S. aureus. The thiolated aptamer exhibited specific binding affinity towards S. aureus, whereas AuNPs with the unbound aptamer underwent varying degrees of aggregation following freeze–thaw, leading to distinct alterations in color. After the aggregation of AuNPs, FRET between AuNPs and FITC diminished, followed by a gradual restoration of fluorescence. Under optimal experimental conditions, the colorimetric mode exhibited a detection limit of 5 CFU/mL for S. aureus, while the fluorescence mode demonstrated a detection limit of 2 CFU/mL. The validity of the developed method was confirmed through the plate counting method, with no significant disparities observed in the obtained results. Compared to the plate culture method, the proposed dual-mode strategy can be completed within 95 min, greatly shortening the detection time. Consequently, this method holds promising potential for the rapid and sensitive analysis of S. aureus.

Intelligent design and construction of novel APN-based theranostic probe driven by advanced computational methods

Multifunctional molecules with both optical signal and pharmacological activity play an important role in drug development, disease diagnosis, and basic theoretical research. Aminopeptidase N (APN), as a representative tumor biomarker with anti-tumor potential, still lacks a high-precision theranostic probe specifically targeting it. In this study, a novel quaternity design strategy for APN theranostic probe was developed. This proposed strategy utilizes advanced machine learning and molecular dynamics simulations, and cleverly employs the strategy of conformation-induced fluorescence recovery to achieve multi-objective optimization and integration of functional fragments. Through this strategy, a unique “Off–On” theranostic probe, ABTP-DPTB, was ingeniously constructed to light up APN through fluorescence restoration, relying on conformation-induced effects and solvent restriction. Differ from the common diagnostic probes, the intelligent design with non-substrated linkage makes ABTP-DPTB for long-term in-situ imaging. The fabricated probe was used for detecting and inhibiting APN in various environments, with a better in vitro inhibitory than golden-standard drug bestatin.

In Situ Cancer-Cell-Triggered Visible Changes in Mechanical Properties, Electroconductivity, and Adhesiveness of a MnO2@PD-Based Mineralized Hydrogel.

Herein, a cancer-specific dopamine-conjugated sp2-rich carbonized polymer dot (PD)-encapsulated mesoporous MnO2 (MnO2@PD)-mineralized hydrogel biosensor was developed that offers cancer-induced observable in situ alterations in fluorescence (FL), electrochemical, and mechanophysical properties. Cancer-triggered MnO2 degradation in the hydrogel, prompted by increased levels of glutathione (GSH) and reactive oxygen species (ROS) such as H2O2, leads to PD release and FL restoration, thereby controlling changes in the pore structure and increasing hydrogen bonding, resulting in physiologically visible alterations in mechanical stretchability, viscosity, swelling behavior, and adhesiveness. The pore size of the matrix increased from 21.83 to 36.81 m2/g upon GSH treatment, affecting the viscosity and swellability of the system. The resistance increased from 21.96 ± 1.16 to 30.69 ± 2.01 and 32.21 ± 2.54 kΩ, respectively, confirming the dependence of conductivity changes on H2O2 and GSH treatments. The in vitro treatment with cancer cells (HeLa, PC-3, and B16F10) facilitated a tunable electrochemical sensing performance via redox-mediated MnO2 breakdown by intracellular ROS and GSH, whereas hydrogels treated with normal cells (CHO-K1) showed minimal changes. Cancer-microenvironment-derived water-drop sensing showed three times higher response as compared to the normal cell-treated hydrogel. The sensing capability of the fabricated sensor was validated based on bending-induced relative resistance changes under dry and wet conditions. Moreover, the integration of the developed sensor with a wireless sensor enabled real-time monitoring with a smartphone.

Nano-fluorescent quantum dots as substrates for determination of ribavirin in pharmaceuticals and human plasma as well as monitoring of its kinetic interaction with salmon sperm DNA

Ribavirin (RIB) was successfully determined by fluorescence spectroscopy upon its quenching to environment friendly phosphorus and nitrogen co-doped carbon quantum dots (PNQDs). Different analytical parameters affecting the fluorescence spectra have been optimized and validated in accordance to the ICH guidelines. The proposed method has provided an efficient tracing of the interaction between RIB molecules and the synthesized QDs in an acidic medium (off-mode). The RIB molecules have shown excellent sensitivity by quenching of the emission band at 401 nm upon excitation at 245 nm throughout a linear range of 0.06–10.00 µg/mL with detection and quantitation limits down to 14.00 and 40.00 ng/mL, respectively. The quenching mode was proven to be static in raw samples and samples extracted of spiked plasma for quenching rate constants of 1.30 × 1012 L M−1 S−1 and 1.73 × 1012 L M−1 S−1, respectively. The proposed method has been successfully applied for determination of RIB in the commercial capsules and spiked human plasma samples with good recovery percentages in between 102.00 and 103.00%. Interestingly, these carbon dots have been utilized as nano-fluorescent platforms for assessment of the binding interaction kinetics between the RIB molecules and salmon sperm DNA (ssDNA). This has been implemented through peeling off the RIB molecules from surface of the PNQDs upon successive addition of the ssDNA and hence fluorescence restoration (turning on). Consequently, this provides a successful monitoring of its antimicrobial potency. It was evidenced a strong binding interaction with a binding constant of 2.38 × 104 mol−1/L. Significantly, this could open doors for an extended application for on-site monitoring of RIB as well as its interactions with biomolecules and microorganisms.

Electrochemical and fluorescent dual-mode sensor of acetylcholinesterase activity and inhibition based on MnO2@PD-coated surface

We developed an electrochemical and fluorescent dual-mode sensor for assessing acetylcholinesterase (AChE) activity and inhibition by taking advantage of the high redox sensitivity of surface-coated mesoporous MnO2@polymer dot (MnO2@PD) towards AChE. The following phenomena constitute the basis of the detection mechanism: fluorescence resonance energy transfer (FRET) effect between MnO2 and PD; catalytic hydrolysis of acetylthiocholine (ATCh) to thiocholine (TCh) by AChE expressed by PC-12 cells, inducing fluorescence restoration and change in the conductivity of the system due to MnO2 decomposition; the presence of the inhibitor neostigmine preventing the conversion of ATCh to TCh. The surface-coated biosensor presents both fluorescence-based and electrochemical approaches for effectively monitoring AChE activity and inhibition. The fluorescence approach is based on the fluorescent “on/off” property of the system caused by MnO2 breakdown after interaction with TCh and the subsequent release of PDs. The conductivity of the coated electrode decreased dramatically as AChE concentration increased, resulting in electrochemical sensing of AChE activity and inhibition screening. Real-time wireless sensing can be conducted using a smartphone to monitor the resistance change, investigating the potential use of MnO2@PD nanocomposites in biological studies, and offering a real-time redox-fluorescent test for AChE activity monitoring and inhibitor screening.

Proximity binding-initiated DNA walker and CRISPR/Cas12a reaction for dual signal amplification detection of thrombin

We report here a highly sensitive fluorescent thrombin biomarker sensing method by integrating the DNA walker and CRISPR/Cas12a system. The presence of thrombin causes the localization of DNA moving arms on AuNP tracks via their proximity bindings with the dye-labeled probes immobilized on AuNPs. With the assistance of the primer and DNA polymerase, the arm sequences move continuously on the AuNP tracks to generate many CRISPR/Cas12a-responsive dsDNAs, which push the dye to move away from AuNPs to restore its fluorescence. Moreover, the dsDNAs can be recognized and cut by the CRISPR/Cas12a to trigger its trans-cleavage activity for cyclically cleaving the fluorescently quenched signal probes on the AuNP tracks, which liberates the dye from AuNPs to further enhance the fluorescence restoration to achieve highly sensitive thrombin assay with detection limit of 29.5 fM. Selectively detecting thrombin against other interference proteins and in diluted serums by such sensing method has also been verified, making it an attractive approach for monitoring other protein biomarkers at low levels for the diagnosis of diseases.